This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project V: Robinson (To be discontinued for June 2006) Integral membrane proteins play critical roles in cell function, and are targets for a majority of drugs on the market and in development. However, understanding of their structures and functions and our ability to produce and study them lag far behind soluble proteins. Little is known about the forces and principles governing stability and assembly for integral membrane proteins. Our goal is to identify key determinants of protein stability, pathways for the folding and assembly of a member of the G-protein-coupled receptor superfamily. We intend to measure mutational effects and side-chain interactions in native, intermediate, and inactive states of the receptor, to identify contacts that direct folding and misfolding. We will use intrinsic fluorescence to monitor environments of specific domains during folding. In parallel studies we are characterizing the helical propensity, intrinsic stabilities, and association of the seven helices to develop a model for helix formation and association during folding. These studies will increase understanding of membrane protein folding, and lead to more effective methods for production of these valuable proteins for structural studies, biochemical analysis, and drug discovery application. Substantial progress has been made in identifying an intermediate in the folding pathway of the A2a receptor, and characterized an off-pathway inactive state. We are initiating mutagenesis studies to elucidate the structural contacts present in each state. We have shown that peptides corresponding the 7 TM helices of A2a receptor are helical, and are studying their interactions, to enable us to develop a model for the folding pathway.